WO2003083479A1 - Blood processing method, blood processing device, method of measuring hemoglobins and device for measuring hemoglobins - Google Patents

Abstract

A blood processing method involving the step St1 of preparing a blood specimen sample and a hemolytic agent and the step St2 of preparing a mixture sample by mixing the blood specimen sample with the hemolytic agent, as shown in Fig. 1. The hemolytic agent to be used herein is a solid drug capable of breaking erythrocyte membrane.

Description

 Meishinda Blood processing method, blood processing device, hemoglobin measurement method and hemoglobin measurement device

 The present invention relates to a blood processing method for destroying red blood cell membranes and measurement of hemoglobins. Background art

 At present, point 'ob' care 'testing (hereinafter abbreviated as PO CT) is attracting attention in the field of clinical examination. POCT refers to a clinical examination in a medical field, and refers to a general term for an examination where the time from sample collection to the output of the test is the most important. Therefore, in general, in the field of clinical examination, ease of operation, quick judgment, and low price are required. This also applies to the measurement of blood constituents.

 Blood is roughly divided into cellular components and liquid components. Cellular components include red blood cells, white blood cells, and platelets. The liquid component includes plasma.

 In blood sample samples, the volume ratio of red blood cells to whole blood (hereinafter referred to as hematocrit or Ht value) is one of the important factors. The Ht value changes depending on the degree of anemia, so it is a measure to know the degree of anemia. The Ht value is also a measure of the need to make corrections to other measurements, as physical properties (such as viscosity) of the blood sample may affect the measurement.

Erythrocytes are about 1. 1 x 10 ^{1 Q} and occupy a volume of 1 ml, and the volume per piece is about 90 〃m ³ . Water, the largest component of erythrocytes, accounts for about 63 g in 100 ml of erythrocytes. Most of the remaining approximately 34 g of red blood cells can be regarded as hemoglobins. When the Ht value is 45%, the amount of hemoglobin in 1 L of blood is about 150 g. That is, hemoglobins are present in the blood at a concentration of 150 g / 1 and are the most abundant proteins in the body.

Human hemoglobin (hereinafter referred to as Hb) is a chain of non-chains (?,?, (5)). These globins are tetramers associated with heme and associated with heme. The healthy individuals Hb, H b A (shed ₂ 2?) Of about _{9 0%, H b Α 2} (α 2 δ 2) about 3%, H b F (0 : 272) is about 1%, And HbA, (S-linked HbA) are present in several% respectively. The above H bA, t is, HbA, _a, HbA _lb, and H b A,. Is included.

Conventionally, examination of the composition ratio of H b is considered to be important in clinical chemistry. For example, if the measured value of Hb F indicates an abnormal value other than the standard range of 0.3 to 1.3%, suspicion of hereditary hyper Hb F disease, chronic myelogenous leukemia, acute myelogenous leukemia, etc. is suspected. . In addition, when the measured value of H bA ₂ shows an abnormal value other than the reference range of 2 to 3.5%, unstable Hb disease or malignant anemia is suspected.

Furthermore, diabetes, which is one of the three major adult diseases, has become a problem in recent years. Therefore, Hb Ai or H bA _lc is attracting attention as a test item as a guideline for relatively long-term glycemic control of diabetic patients for 1 to 3 months. A measuring device for H bA _lc has been developed.

 Hemoglobins are measured, for example, using isoelectric focusing, ion exchange chromatography 1, latex immunoagglutination, and the like.

Unlike other analytes in blood samples, hemoglobins are present in red blood cells, so hemolysis needs to occur in order to make measurements. Hemolysis means that the erythrocyte membrane is broken and hemoglobins are excreted out of the erythrocytes. _C More specifically, the size of the erythrocytes is affected by the osmotic pressure to the erythrocyte membranes. In a salt solution whose salt concentration is higher than that of normal saline (0.9% NaC1), red blood cells contract. On the other hand, red blood cells expand in a salt solution whose salt concentration is lower than that of saline (0.9% NaC1). In the process of contraction and expansion of such red blood cells, when the red blood cell membrane is broken, hemoglobins are excreted from the red blood cells. If the blood is completely hemolyzed, and the H t value is 45%, the hemoglobin concentration is about 150 g / L. Problem to solve

The results of measurement of hemoglobins are shown as a percentage of total hemoglobin. Therefore, not only the amount of hemoglobin to be measured but also the total amount of hemoglobin is measured. Need to As described above, the total amount of hemoglobin is usually present in blood at a concentration of about 150 g / L although there are individual differences. This concentration is a very high concentration which is inconvenient for the measurement of hemoglobins. Since hemoglobins have a red dye having an absorption wavelength on the long wavelength side, measurement of hemoglobins is likely to be disturbed at high concentrations.

 For example, when measuring hemoglobins by immunochromatography, a decrease in the measurement value in the antigen excess region that occurs as one characteristic of the immune reaction causes an erroneous operation. In addition, the increase in the viscosity of the blood sample sample due to a large amount of hemoglobins causes a problem such as the development speed of the blood sample on the solid phase matrix of the immunochromatography-one device being slowed down or not expanding at all. Therefore, for example, as disclosed in JP-A-3-46566, in order to accurately measure hemoglobins, many operations for diluting a blood sample after hemolysis are necessary. is necessary. That is, in order to measure hemoglobins, many operations for processing blood sample samples are required.

 The present invention has been made in view of the above circumstances, and relates to a blood processing method, a blood processing device, a method for measuring hemoglobins and a device for measuring hemoglobins, and in particular, a component of red blood cells represented by hemoglobins. The purpose is to provide a method and device that can make the measurement easier. Disclosure of the invention

 The blood processing method of the present invention comprises the steps of: preparing a blood sample containing red blood cells; and a hemolytic agent; and (b) preparing a mixed sample by mixing the blood analyte sample and the hemolytic agent. b) and the above-mentioned hemolytic agent is a drug which destroys the erythrocyte membrane which is solid.

In the conventional method, in order to measure components in red blood cells represented by hemoglobins, many operations for processing a blood sample are required. However, when using the blood processing method of the present invention, when measuring components in red blood cells, many operations for diluting a blood sample after hemolysis are unnecessary. Therefore, according to the blood processing method of the present invention, the components in red blood cells can be measured more easily. O

 In the step (b), it is preferable that blood cell components remain in the mixed sample.

 The hemolytic agent may be a drug that changes the osmotic pressure to the erythrocyte membrane.

 The hemolytic agent may be configured as an inorganic salt.

 The concentration of the inorganic salt in the mixed sample is preferably in the range of 0.5 to 10% (w / v).

 The method for measuring hemoglobins according to the present invention comprises the steps of: (a) preparing a blood sample containing red blood cells; and a hemolytic agent; and (b) preparing a mixed sample by mixing the blood specimen and hemolytic agent. And a step (c) of measuring hemoglobins contained in plasma in the mixed sample, wherein the hemolytic agent is a drug which is a solid that destroys an erythrocyte membrane.

 The conventional method requires many operations to process a blood sample to measure hemoglobins. However, when using the method for measuring hemoglobins of the present invention, when measuring hemoglobins, many operations for diluting a blood sample after hemolysis are unnecessary. Therefore, according to the hemoglobin type measuring method of the present invention, measurement of hemoglobins can be performed more easily. In the step (c), as a method of measuring the hemoglobins contained in the plasma in the mixed sample, an immunochromatography method, an immunoconcentration method, an enzyme-linked immunosorbent assay method, a colorimetric slide method, and Any one of the electrode method sliding method may be used.

 In the step (c), at least two types of hemoglobin may be measured.

 The at least two types of hemoglobin may be configured to be a combination of hemoglobin and H b A 1 c.

 The hemoglobin may be glycated hemoglobin.

The blood processing device of the present invention comprises: a substrate; and a solid hemolytic agent for destroying the erythrocyte membrane, wherein the hemolytic agent is a mixture of a blood sample containing erythrocytes and the hemolytic agent. An amount of the blood cell component remaining in the mixed sample is supported on the base when the fuel is formed.

 According to the blood processing device of the present invention, it is possible to more easily measure the components in red blood cells.

 The hemoglobins measuring device of the present invention is a hemoglobins measuring device for measuring hemoglobins in a blood sample containing red blood cells, comprising: a substrate having the above-mentioned addition part of the blood sample, A hemolytic agent for destroying the erythrocyte membrane, a detection substance that specifically binds to the hemoglobin, and an immobilization substance that specifically binds to the hemoglobin; An amount of the blood cell component remaining in the mixed sample is supported on the base when the mixed sample of the hemolytic agent and the hemolytic agent is formed, the hemolytic agent, the detecting substance, and the detecting substance. The immobilized substance is solid, and is supported on the base material, and the hemolytic agent, the detection substance, and the immobilized substance in the order from the side closer to the addition portion of the blood sample of the base material. Is placed o

 According to the hemoglobin type measuring device of the present invention, measurement of hemoglobin types can be performed more easily.

 Another hemoglobins measuring device according to the present invention is a hemoglobins measuring device for measuring hemoglobins in a blood sample containing red blood cells, comprising: a substrate; and the above blood sample provided on the substrate A sample addition part, a solid hemolytic agent provided on the substrate, for destroying a red blood cell membrane, a hemolytic layer for controlling hemolysis of the blood sample, provided on the substrate, A detection layer which specifically binds to the hemoglobins, and a labeling layer for labeling the hemoglobins, and an immobilization reagent which is provided on the substrate and which specifically binds to the hemoglobins, And a detection layer for detecting the hemoglobins, wherein the blood sample can move between the hemolysis layer, the labeling layer, and the detection layer.

 According to the hemoglobin type measuring device of the present invention, measurement of hemoglobin types can be performed more easily.

The blood sample sample may be configured to further include a layer for removing red blood cell residue after hemolysis. The hemolytic layer, the recognition layer, and the detection layer may be arranged in a single layer. Brief description of the drawings

 FIG. 1 is a flow chart of the blood processing method of the present invention.

 FIG. 2 is a diagram showing a formula representing the hemolytic rate.

 Fig. 3 (a) and Fig. 3 (b) show the blood processing device of this embodiment o

 FIG. 4 is a view showing an example of a single-layer type hemoglobins measuring device according to the present invention c FIG. 5 is a cross-sectional view showing an example of a multi-layer type hemoglobins measuring device according to the present invention.

 FIG. 6 is a diagram showing the concentration of KC 1 (added amount of a hemolytic agent) and the light transmittance in one example of the present invention.

 FIG. 7 is a diagram showing the relationship between the concentration of KC 1 (added amount of a hemolytic agent), the Hb concentration and HbA 1 c in one example of the present invention.

 FIG. 8 is a graph showing the relationship between the concentration of KC 1 (the addition amount of a hemolytic agent) and the reflection absorbance at 610 nm in one example of the present invention.

 FIG. 9 is a diagram showing a calibration curve of the device for Hb measurement in one embodiment of the present invention o

 FIG. 10 is a diagram showing a calibration curve of the device for HbA measurement in an example of the present invention.

FIG. 11 is a diagram showing the results of measuring 4.0% Hb A,., 5. 4% Hb A 10. 4% Hb A _le in the device according to one embodiment of the present invention.

FIG. 12 shows the results of concentration conversion using the calibration curve from the measured values of 4.0% HbA, o, 5.4% Hb Ac, and 10.4% HbA _le by the device according to one example of the present invention. FIG. Best embodiment

Hereinafter, embodiments of the present invention will be described with reference to the drawings. (Embodiment 1)

 In the present embodiment, the blood processing method of the present invention will be described with reference to the drawings. FIG. 1 is a flow chart of the blood processing method of the present invention.

 As shown in FIG. 1, in the blood processing method of the present embodiment, a mixed sample is prepared by mixing a blood sample, a hemolytic agent, and a step St1; and a blood analyte sample and a hemolytic agent. Step St 2 is included. Here, the hemolytic agent is an agent that is solid and that destroys the erythrocyte membrane.

 A "blood sample sample" is blood collected from a subject, including a human, by a conventional blood collection method (eg, by intravenous injection or lancet) or blood stored prior to measurement.

 As the hemolytic agent, one that is a solid, which is an agent that disrupts the erythrocyte membrane is used. For example, it is preferable to be prepared in powder form so as to be substantially free of water by lyophilization and the like.

 In addition, the blood processing method of the present embodiment is suitably used for measurement of hemoglobins. That is, in the method for measuring hemoglobins of the present invention, as shown in FIG. 1, a step of preparing a blood sample and a hemolytic agent, St 1 and a blood sample and hemolytic agent are mixed. In addition to the step of preparing the mixed sample S t 2, the method further includes the step of measuring hemoglobins contained in plasma in the mixed sample. Here, the hemolytic agent is a solid, which is an agent that destroys the erythrocyte membrane.

 The moist mouth bins contained in the plasma in the mixed sample by the blood processing method of the present embodiment described above can be quantitatively measured by methods well known to those skilled in the art.

 As shown in FIG. 1, when a mixed sample is prepared by mixing a blood sample and a hemolyzing agent in step St2, the red blood cell membrane of red blood cells in the blood sample is broken, and the components in red blood cells (typical Hemoglobins are excreted out of red blood cells (that is, hemolysis occurs). Therefore, it becomes possible to measure components (typically hemoglobins) in red blood cells.

As described above, in order to measure components in red blood cells represented by hemoglobins, many operations for processing a blood sample are required. However, if the blood processing method of the present embodiment is used, when measuring the components in red blood cells, There is no need to perform many operations to get blood samples after Therefore, according to the blood processing method of the present embodiment, the components in red blood cells can be measured more easily.

When measuring hemoglobins by an immunological method, since complete hemolysis is at a very high concentration, it may enter an antigen excess region and reduce the sensitivity of the measurement. _C Therefore, mixing in step St 2 It is preferable that blood cell components remain in the sample, that is, partially hemolyze the blood sample.

 "To partially hemolyze a blood sample sample" means to lyse a volume of red blood cells less than complete hemolysis. For example, in the case of 1 L of blood having an Ht value of 45%, it means to hemolyze red blood cells having a volume of 45 Om or less. In contrast, "complete hemolysis" refers to hemolysis of red blood cells in substantially the entire red blood cell volume. For example, in the case of 1 L of 111% 45% blood, it means to hemolyze about 45 Oml red blood cells. By controlling the blood sample sample to be partially hemolyzed, the membrane of some red blood cells in the sample is broken, and hemoglobins in the sample can be eluted. At this time, the concentration of hemoglobins in plasma is lower than in the case of complete hemolysis. For example, when 1 L of blood having an Ht value of 45% hemolyzes a red blood cell equivalent to a volume of 1 ml, 0.34 g of hemoglobins and 0.63 g of water are eluted in 55 O ml of plasma. Therefore, 0.34 g of hemoglobin is dissolved in 550.63 ml of plasma water, so the concentration of hemoglobin is 0.62 g / L. The total hemoglobin concentration when completely hemolyzed is 150 g / L. According to the blood processing method of the present embodiment, an effect equivalent to about 250-fold dilution can be obtained as compared with complete hemolysis, only by hemolysis, without requiring a buffer solution or the like.

As mentioned in the background section, the H t value is the volume ratio of red blood cells to whole blood. For this reason, the amount of red blood cells and the total amount of hemoglobin can be understood from the Ht value. The Ht value is determined by well-known methods including capillary method and Wint robe method. The Ht value may be 36-48% male and 34-43% female in adults as determined by capillary method. In infants, neonatal (cord blood) 44-64%, 3 months 32-44%, 1-year-old children 36-44%, 10- 12 years 37-44% (by Dacie et al .; Clinical laboratory recommendations, revised 31st edition, Kinbara Publishing Co., Ltd. See for reference). Therefore, the Ht value is set for hemoglobin measurement taking into account the sex and age of the subject.

 The total hemoglobin concentration varies with individuals such as H t value, but most people have Hb more than 10 Omg / ml (Male: about 130 to 170 mg / m 1, Female: about 120 to 15 Omg / m ml). Conventional measurement instruments can not measure Hb at concentrations greater than 10 Omg / ml without performing dilution procedures. Therefore, the Hb concentration by "partial hemolysis" is preferably a concentration of 10 Omg / ml or less.

Control of hemolysis can be performed, for example, by changing the amount of hemolytic agent. For example, when 1.5 ml of blood is added to 1.5 mg or 7.5 mg of potassium chloride, the total hemoglobin concentration is about 25 g ZL for the former and about 6.0 g / L _{s for the} latter. Thus, as the amount of hemolytic agent increases, the rate of hemolysis also increases. Therefore, we briefly describe the determination of the amount of hemolytic agent that achieves the optimal hemoglobin concentration (optimum hemolysis rate) in plasma.

 First, the blood sample is hemolyzed with an arbitrary amount of hemolyzing agent, and the supernatant fluid is removed by separation. Next, measure the concentration of hemoglobin in the supernatant fluid. Subsequently, the relationship between the addition of the hemolytic agent and the hemoglobin concentration is determined. Finally, the addition amount of the hemolyzing agent to achieve the optimum hemoglobin concentration in plasma is determined based on the above relationship.

 In detail, the procedure is carried out by obtaining the relationship between the amount of the hemolytic agent and the hemoglobin concentration by performing a procedure substantially similar to the procedure described in Example 2 described later. Since the optimal hemoglobin concentration (optimum hemolysis rate) in plasma depends on the method of measurement, the amount of hemolytic agent added also depends on the method of measurement. In the present specification, the hemolysis rate is represented by the formula shown in FIG.

 When the Ht value is 40 to 50%, and the measurement method of hemoglobins is absorption method, the hemolytic agent has a hemolysis rate of 0.10 to 1% (hemoglobin concentration 0.10 to 1: Lmg / m 1) It is preferable to add such an amount, and when the measurement method is an immunochromatography method, the hemolysis rate is 0.10 to 0.1% (hemoglobin concentration 0.1 to 1 Omg / d 1) Preferably it is added in such an amount.

As measured by absorption spectroscopy, the maximum molar extinction coefficient of Hb is approximately 1 Q 5 M ^-1 , The amount of addition of the hemolytic agent is preferably such that the hemoglobin concentration is 0.1 to 1 mg / ml (the concentration at which the absorbance is about 0.1 to 2).

 As determined by immunochromatography, the amount of hemolytic agent added is based on the concentration of hemoglobin which is dependent on the performance (such as binding affinity) of the antibody used in the immunographography method.

 For example, when potassium chloride is used as the hemolytic agent, generally, the addition ratio (concentration) of the hemolytic agent to the total volume of the blood sample is 0.05% to 10% (w / v) (herein, The unit of w / v) should be g / ml. In particular, the addition ratio (concentration) of the hemolytic agent to the total volume of the blood sample is in the range of 0.5 to 1 to 1% (w / v) in the case of the absorption method, and in the case of the immunograph oral method Although depending on the size of the chromatography device, or the amount of the immobilized antibody carried on the chromatography device, and the reagent amount of the labeled antibody to some extent, it is preferable to be within the range of 0.5 to 3% (w / v). In particular, in the case of a 5 mm × 10 cm chromatography device, it is preferable to be in the range of 0.1 to 1% (w / v).

 When other salts are used, they may be added at a concentration equivalent to that of chlorination. Also, detergents such as, for example, sodium laurate, or agents which act directly on the erythrocyte membrane, such as anti-red blood cell antibodies, can also be used. Also in this case, after determining the amount of hemolytic agent necessary to achieve the optimal hemoglobin concentration in plasma using the above-mentioned method of measuring the total hemoglobin concentration, the determined amount of hemolytic agent is determined. The blood sample may be partially hemolyzed using

Once the amount of hemolytic agent necessary to achieve optimal hemoglobin concentration in the plasma is determined, this amount of hemolytic agent will be controlled to partially hemolyze the blood sample sample. used. Then, any hemoglobin assay currently used in clinical testing is used to measure total hemoglobin concentration in blood sample samples. Such hemoglobin determination methods include, for example, the cyanomethemoglobin method, the oxyhemoglobin method, and the SLS-hemoglobin method (see, for example, clinical test method presentation, revised 31st edition, Kinbara Publishing Co., Ltd.). ). Hemogro mentioned above Dedicated in vitro diagnostic agents used in bottle determination are commercially available.

 According to the blood processing method and the hemoglobins measuring method of the present embodiment, when hemoglobins are measured, many operations for diluting a blood sample sample after the hemolysis operation conventionally required are omitted. Therefore, it is possible to easily realize an appropriate hemoglobin concentration for measurement.

 In the present embodiment, an agent that changes the osmotic pressure to the erythrocyte membrane is used as the hemolytic agent. Erythrocytes contract in a salt solution whose salt concentration is higher than that of saline (0.9% NaC1). On the other hand, red blood cells swell in a salt solution whose salt concentration is lower than that of saline (0.9% NaC1). Such contraction and expansion of red blood cells break up the red blood cell membrane, and hemoglobins are excreted from the red blood cells. For example, inorganic salts (eg, potassium chloride, sodium chloride or sodium fluoride etc.) can be mentioned. In particular, potassium chloride is preferred. As described above, hemolysis occurs when erythrocytes are placed in an environment other than the physiological condition of 0.9% Na C1. Therefore, the above-mentioned drug is treated under such conditions (that is, concentration excluding salt concentration of 0.9% Na C 1 or equivalent) in the blood, and desired. An amount of hemoglobin is added to the blood sample at a concentration or amount such that it elutes into plasma. For example, using 250 / g of potassium chloride for a blood volume of 501, the hemolysis rate is about 2%.

 The hemolytic agent is used in solid form. As a hemolytic agent, a solution containing a desired amount of a hemolytic reagent may be prepared, and the solution may be prepared by lyophilization. Alternatively, for example, a solution containing a desired amount of a hemolytic reagent may be impregnated with a substrate (such as membrane) and then freeze-dried to use a solution supported on the substrate.

Blood sample samples are treated with a hemolytic agent before the measurement of hemoglobins is initiated. Alternatively, a blood sample is treated with a hemolytic agent simultaneously with the measurement of hemoglobins. In any event, the hemolysed hemoglobins are measured by means or techniques that can be used in the art, as detailed below. As an embodiment of such a method, when the hemolytic agent is in a solution state, the hemolytic agent can be added to the blood analyte sample prior to the measurement of hemoglobins in the blood analyte sample. Hemolysis When the agent is solid and supported on a substrate, a blood sample can be provided to the measuring means after being introduced to the substrate, or when such a substrate is provided to the measuring means, A charge may be introduced to the substrate.

Hemoglobins to be measured include Hb, Hb A, 0ίιβ 2, Η b ( ₂ ( _{2 2} ζ ζ), Hb F ( ₂ァ, HbA! (HbA and a conjugate of sugar), and H b is further classified, HbA _la, Ru include H b Aibs and H b a! _c. here, Hb a, that means the total hemoglobin including the above-mentioned hemoglobin. Hb at and the HbA! HbA _la , HbA _lb , and HbA _lc are classified as glycohemoglobin or "glycated hemoglobin" and have the same primary structure, but the amino group of the N-terminal palin of the? The term “glycated hemoglobin” as used herein is H b A, as well as the H b A, further classified as Hb A _la , Hb A _lb , and Hb A _lc . encompasses. in particular, HbA _lc is glucose bound non-enzymatically Is a hemoglobin to reduction.

 Examination of the compositional ratio of Hb is clinically important. For clinical significance, at least two hemoglobins can also be measured in a blood sample treated as described above.

Particularly in the present embodiment, by controlling partial hemolysis, the concentration of Hb (total hemoglobin) in the blood sample can be reduced to a low concentration. For this reason, the concentrations of hemoglobins measured are not absolute values. Table That, HbA, HbA _2, HbF, Hb A and Hb Ai sugar is bonded, HbA, but still classified HbA _la, HbA _lb, measured values such as our and Hb A _lc is a percentage of the total amount of Hb Be done. And since the ratio to these total Hb amounts does not change with the hemolysis rate, it may be sufficient for the measurement object of the present invention. In addition, since the absolute value of hemoglobin concentration is correlated with the hemolysis rate, it is also possible to calculate back from the measured value.

Glycated hemoglobin (in particular, HbA or HbA _lc ) is noted as an indicator of glycemic control in diabetic patients, and more specifically, as an indicator of long-term (mainly 1 to 3 months) blood glucose control in diabetic patients. There is. For example, HbA, if normal, is Hb (total hemoglobin)? ~ 8%, HbA _lc is normal, Hb (all 6% or less (eg, 4. 7 to 5. 7%) of hemoglobin). However, these glycated hemoglobins are increased in diabetic patients. For example, in the case of HbA _lc , it extends to 20% of Hb. Therefore, the measurement method of the present embodiment can be suitably used for diagnosis, treatment and the like of diabetes by measuring glycated hemoglobin. In addition, Hb (total hemoglobin) and HbA _lc can also be measured by the measurement method of the present invention.

 Moreover, according to the measurement method of the present embodiment, HbF can also be measured as a ratio to Hb. The ratio of HbF to Hb is 0.3-3. 3% in normal adults. The ratio of HbF to Hb is usually: hereditary hyperfetal hemoglobinemia, chronic myelogenous leukemia, Fanconi anemia, erythroleukemia, paroxysmal nocturnal hemoglobinuria, refractory anemia, pregnancy, alveolar mole, Thalassemia (?, (5?)), Unstable hemoglobinosis, rarely aplastic anemia, leukemia, polycythemia (erythrocytosis), myelofibrosis, malignancy, thyroiditis, this reference range Therefore, the measurement method of the present embodiment can also be used to diagnose the above-mentioned diseases.

Furthermore, according to the measurement method of the present embodiment, HbA ₂ can also be measured as a ratio to Hb. 11 proportion of pictmap eight ₂ for 111) in the case of a normal person, a 2 to 3.5%. The ratio of HbA _{2 to} Hb is? Thalassemia, unstable hemoglobinosis, sickle cell anemia heterozygote, megaloblastic anemia, hyperthyroidism, which is higher than the reference range, while 、-, 5-, 65- thalassemia, heritability Decreased from the standard range with hyperfetohemoglobinemia, iron deficiency anemia, and ironoblastic anemia. Therefore, the measurement method of this embodiment can also be used to diagnose the above-mentioned diseases.

The above Hb composition components can be quantitatively fractionated by ion exchange chromatography because they have different isoelectric points. Ion exchange chromatography, for example, although C YPRE SS Co. columns may be carried out using (trade name P 01 y CATA ^TM) available from, available commercially as long as it has a fractionation capacity similar to the column Any column can be used. The above-mentioned Hb composition components can also be measured using immunological methods, using an antibody that can individually recognize these components. Immunological methods include, for example, immunochromatography, immunoconcentration, solid-phase enzyme-linked immunosorbent assay (EL ISA), latex agglutination Immunization methods etc. may be mentioned.

 The concentration of total hemoglobin can, as mentioned above, be any hemoglobin assay currently used in clinical examination. As such a hemoglobin assay method, for example, a cyanomethemoglobin method, an oxygenated hemoglobin method, and an SLS-hemoglobin method can be mentioned (for example, clinical test method provision, revised 31st edition, Kinbara Publishing Co., Ltd. See for reference). Dedicated in vitro diagnostic agents used in the above-described hemoglobin assay are commercially available.

 In addition, measurement of hemoglobins is performed by the method of measurement by dry chemistry. The “method of measurement by dry chemistry” means that a liquid sample is added (usually by spotting) to a solid phase matrix on which the reagent is supported in a dry state, and the reagent is reacted with the reagent, and It refers to a method of measuring a test substance in a sample by detecting a reaction. Because dry chemistry measurement methods are quick, simple, and accurate, they are conveniently used in routine clinical examinations. Methods of measurement by dry chemistry include, for example, immunochromatography methods, immunoconcentration methods, immunological methods such as solid phase enzyme immunoassay methods, colorimetric slide methods, and electrode method slide methods. . Dry chemistry is disclosed, for example, in “Clinical Pathology, Dry Chemistry-A New Development of Simple Tests (Edited by the Japanese Society of Clinical Pathology, published in January 1997)”.

(Embodiment 2)

In this embodiment, a blood processing device that can be used for the blood processing method and the hemoglobins measuring method described in the above embodiment 1 will be described with reference to the drawings. FIG. 3 (a) and FIG. 3 (b) FIG. 3 is a view showing the blood processing device of the present embodiment c. As shown in FIG. 3 (a), the blood processing device 10 a of the present embodiment includes: a substrate 11 on which a blood sample is provided; 11. A hemolyzing agent (not shown) in an amount for partially hemolyzing a blood sample, carried on one of the cells. The hemolytic agent is a solid agent that destroys the erythrocyte membrane. The hemolytic agent is first added in a solution state to the substrate, then freeze-dried and carried on the substrate in a dry state. Preferably, the hemolytic agent is a substrate The blood sample is carried in a dry state in the area where it can be contacted.

 As a substrate 11 on which a blood sample is to be provided, a flat plate (such as insoluble single layer substrate (eg, Ceralyzer 1 (A mes), test paper, immunochromatography Container form (eg, vial, test tube, ampule, Sinotest, etc.) containing the sample liquid (eg, plastic card type) or multilayer substrate (eg, Kakutakem (Kodak), Drychem (Fuji) etc.)) Yuichi, piccolo etc.) can be various. The substrates that may be used in the device of the present embodiment are also paper towels (e.g., pulp non-woven fabrics such as Lead Cooking Paper (R)), mesh fabrics (e.g., gauze cotton cloth), and glass fiber filter paper. It is made using materials that can absorb and hold liquids, such as

 Means for measuring hemoglobins include measuring means based on ion exchange chromatography or immunological methods (eg, latex immunoagglutination).

 Also, in place of the blood processing device 10 a shown in FIG. 3 (a), a blood processing device 10 b shown in FIG. 3 (b) can be used. The substrate 11 used in the blood processing device 10 b has a side and a bottom (not shown), and comprises an opening 13 and is a cell 12 suitable for use in the measuring means. In this case, the area where the hemolyzing agent can contact the blood sample in the cell (for example, the bottom and side surfaces in the cell 12 etc. (these are appropriately selected according to the blood sample sample volume to be measured) ) Is supported in dry condition. The blood processing device 10 b may be introduced into the measuring means after the blood sample is introduced into the cell 12 on which the hemolytic agent is carried, and the cell 12 of the blood processing device 1 O b serves as the measuring means. When introduced, a blood sample may be introduced into the cell 12. Alternatively, a blood sample may be introduced into the cell in a batch or flow manner.

 As described in the Background section, in order to measure hemoglobins, many operations for processing a blood sample are required.

In particular, in the past, automated instruments based on the measurement principle of ion exchange chromatography and latex immunoagglutination have been used to measure H b A _1c . The total amount of hemoglobin, such as about 150 g in blood, is extremely high for measurement with the above-mentioned conventional automated equipment. Therefore, in general, the above-mentioned automated instrument comprises: completely hemolyzing a blood sample, diluting the completely hemolyzed sample to a concentration at which an optimal measurement result can be obtained, and The stage of measurement based on is strictly set. Representative H b A _lc dedicated measuring instruments include A 1 c 2. 2 and DLS 2 0 0 0 the Bayer one made Toso. The former is based on ion exchange chromatography principle and the latter is based on latex immunoagglutination inhibition method. The methods for processing blood samples in these devices are common in that the complete hemolysis and dilution steps are automated. The above-mentioned automation equipment is widely used in the field of clinical examination, but the equipment is large-scale and expensive because it automates various operations.

 Thus, in the measurement of hemoglobins, the use of an automated apparatus in which the above-described processing operation is incorporated in the apparatus is expensive, or when such an apparatus is not used, multistage operation Makes the inspection complicated. Furthermore, when hemoglobins are measured by an immunological method, since the concentration is extremely high when complete hemolysis is performed, it may enter the antigen excess region and the measurement sensitivity may be reduced.

 However, if the blood processing device 10a according to the present embodiment is used, the measurement of the component in the red blood cell can be rapidly started only by providing the blood sample to the substrate 11 and setting it on the measuring means. In addition, if the blood processing device 10 b according to the present embodiment is used, the blood sample can be introduced into the cell 12, and the cell 12 can be simply placed in the measuring means to rapidly measure the components in the red blood cell. It can start.

(Embodiment 3)

 In this embodiment, a device for measuring hemoglobin in a blood sample sample by a measurement method using dry chemistry (hereinafter, referred to as a hemoglobin measurement device) will be described. The hemoglobin measurement device includes: an amount of a hemolyzing agent that partially hemolyzes a blood sample, an elutable detection substance that specifically binds to the hemoglobins, and an immobilization substance that specifically binds to the hemoglobins .

The “partially hemolyzable amount of hemolytic agent” used herein is as described in Embodiment 1 above. That's right.

The detection substance is a substance bound to a label that specifically binds to the hemoglobin to be measured. Such labels are known in the art and include, for example, gold colloids, enzymes (eg, horseradish peroxidase, glucose oxidase), radioactive isotopes (eg, iodine ( ¹²⁵ I) , ¹²¹ I), carbon ⁽¹⁴ c), sulfur ⁽³⁵ S), tritium ⁽³ H), indium ⁽¹¹² I n), and technetium (9 ^9m Tc)), fluorescent labels (e.g., Furuoresein and rhodamine), if Most include piotin. Such a label can also be bound to an antibody against the hemoglobin to be measured to form a detection substance. Any antibody can be used as long as it binds to the hemoglobin to be measured. The antibody may be a polyclonal antibody or a monoclonal antibody. Monoclonal antibodies are preferred.

 The immobilized substance is any substance that specifically binds to the hemoglobin to be measured. Such substances are, when using immunological methods, monoclonal antibodies to the target hemoglobin of the subject. It is preferable that it is a monoclonal antibody that reacts to another epitope than the antibody used in the above-mentioned detection substance. In the hemoglobin measurement device of the present embodiment, the hemolytic agent, the detection substance, and the immobilization substance are carried in a dry state in order from the side closer to the addition site of the blood sample of the device.

 Therefore, according to the hemoglobin measurement device of the present embodiment, the blood sample added to the device moves between the hemolytic agent, the detection substance, and the layer on which the immobilization substance is carried, From elution to detection of eluted hemoglobin can be performed.

In the hemoglobin measurement device of the present embodiment, the hemolytic agent is, for example, impregnated in a layer for supporting on a substrate, and then lyophilized. Since the hemolytic agent reacts upon contact with the added blood, it is preferably contained throughout the substrate so that the site to which the blood is added can be measured without prior determination. Of course, in this case as well, the amount of the hemolytic agent contained in the entire substrate is such that the concentration of the eluted hemoglobins in plasma is lower than that in complete hemolysis. When the blood sample containing the eluted hemoglobins moves to the layer on which the detection substance is supported, the detection substance supported on this layer is eluted, and the eluted hemoglobins are specific to the eluted detection substance and the eluted hemoglobins. React. Thus, the detection substance can be supported in the dry state to form a solid phase when the device is not used, but can be supported on the layer so that it can be eluted in the solution when it is encountered. For example, for loading onto such a layer, the detection substance may be lyophilized after being impregnated into the layer. The hemoglobins bound to the detection substance are bound to the immobilized substance supported on the substrate (solid phase matrix). The amount of hemoglobin in the blood sample is determined by detecting the sandwich-specific reaction of the detection substance, hemoglobin, and the immobilization substance in the solid phase matrix. The solid phase matrix to be detected may be a matrix material (for example, cellulose, agarose, dextran, polystyrene, etc.) which can be used in conventional immunoassay and the like. The solid phase matrix may also be films, test strips and test strips.

 Hemoglobin-based measuring devices based on dry chemistry measuring methods can be single-layer or multi-layer. In either the single-layer system or the multi-layer system, assuming that the one closer to the addition site of the blood sample is the upstream, the hemolytic agent, the detection substance, and the immobilization substance are loaded in this order from upstream to downstream.

 In the single-layer system, as shown in FIG. 4, a layer controlling hemolysis of a blood sample with a hemolytic agent (hereinafter referred to as hemolytic layer in the present specification), a layer labeling hemoglobins with a detection substance (this Hereinafter, in the specification, a label layer) and a layer for detecting hemoglobins bound to the immobilization reagent (hereinafter, also referred to as a detection layer) are contained in a single (identical) layer. Be placed. In the monolayer method, a hemolytic agent, a detection substance, and an immobilization substance are supported on a substrate (solid phase matrix) 41 that can be a place for detection of hemoglobins sequentially from the side A where the blood sample is added. It is done. Solid phase matrix 41 can be a matrix material (eg, nitrocellulose membrane) that can be used in conventional immunoassays and the like. The solid phase matrix can also be films, test strips, and test strips.

With such an arrangement, the added blood sample first contacts the hemolytic agent in the process of spreading on the solid phase matrix to partially hemolyze the blood. next, Hemoglobin in plasma water due to partial hemolysis and elutable detection substance are specifically bound. Subsequently, the complex of hemoglobins-detection substance and the immobilization reagent are specifically bound to produce a detection substance-hemoglobin-immobilization substance.

 In the multi-layer system, the layer that controls the hemolysis of the blood sample by hemolytic agent, the layer that labels hemoglobins by the detection substance, and the layer that detects hemoglobins bound to the immobilization reagent are independent and independent. The layers are laminated in such a manner that the blood sample can move between them. In the multilayer system, the hemolytic agent, the detection substance, and the immobilization substance are carried on separate layers, and the layers are laminated in order from the side to which the blood sample is added. The layer on which the hemolytic agent is supported is made of a material that allows the liquid added to the surface to permeate the opposite surface, such as pulp non-woven fabric, mesh fabric (for example, gauze cotton fabric), and glass fiber filter paper. Using the material, the hemolytic agent produces a layer that controls the hemolysis of the blood sample. The hemolytic agent is carried on the layer, for example, by impregnating these layers with a solution of the hemolytic agent and then freeze-drying. The detection substance can also be carried on a layer made of a material that allows the liquid added to the surface to penetrate the opposite surface. The detection substance is also supported on the layer, for example, by impregnating these layers with a solution of the detection substance and then freeze-drying. The immobilization material can be supported on a solid phase matrix which can be a place of detection of hemoglobins. Such solid phase matrices include nitrocellulose membranes. The immobilization can be carried out by the procedure of immobilization used in conventional immunological assays. These layers can be placed in contact so that blood sample samples can be moved between the layers. With such an arrangement, the added blood sample first contacts the hemolytic agent in the process of moving between these layers to partially hemolyze the blood. Next, the hemoglobin in plasma water by partial hemolysis and the elutable detection substance are specifically bound. Subsequently, the complex of hemoglobins-detecting substance may be specifically bound to the immobilization reagent to produce a detection substance-hemoglobin-immobilizing substance.

The hemoglobin measurement device of the present embodiment may also include a layer that removes red blood cell residue after the blood sample is hemolyzed. The layer that removes this red blood cell residue may be the same as the layer that controls the hemolysis of the blood sample by hemolytic agent. Or this In the device, a layer may be provided which removes red blood cell residue separately from the layer which controls the hemolysis of the blood sample sample by the hemolytic agent. For example, non-woven pulp, mesh cloth

Layers of materials such as gauze cotton fabric and glass fiber filter paper may be used. This layer can absorb plasma solutions containing red blood cell membrane debris that may not be necessary for measurement of hemoglobins by dry chemistry. Therefore, in the present specification, this layer is also referred to as "absorbing layer".

 An example of the multi-layer type hemoglobins measurement device of the present embodiment is shown in FIG. FIG. 5 is a cross-sectional view showing a multi-layer type hemoglobin measurement device according to this embodiment. An antibody-immobilized membrane (22) on which an antibody (23) is immobilized on a substrate (21) (the substrate may be polyethylene terephthalate (PET)) The freeze-dried support layer (24) and then the hemolytic agent support layer on this

(25) can be laminated. Here, as shown in Fig. 2, the hemolytic agent support layer (25), the labeled antibody lyophilization support layer (24), and the antibody-immobilized membrane (22) should partially overlap the layer immediately below. Can be placed. This overlap causes the blood sample sample to move between the layers, thus producing hemolysis, labeled antibody binding, and immobilized antibody binding. An antibody specific for hemoglobin is fixed to the exposed area of the antibody-immobilized membrane (22) (ie, the area where the hemolytic agent-carrying layer (25) and the labeled antibody lyophilization-carrying layer (24) do not overlap). In this area, reactions of hemoglobins in blood sample can be detected. In addition, the absorption layer (26) overlaps the region of the antibody-fixed membrane (22) opposite to the blood sample addition side (that is, the side on which the labeled antibody freeze-drying support layer (24) is not overlapped). Can be stacked as The absorbing layer can absorb the plasma solution containing red blood cell membrane residue, which may be unnecessary for detection, present in the reaction detection area, and can help detect the reaction. Furthermore, a hemolytic agent-supporting layer (25), a labeled antibody lyophilization-supporting layer (24), and an antibody-immobilized membrane

The overlap area of the three layers of (22) to the reaction detection area is impervious to prevent wet drying by the added sample or to avoid contamination of substances that may interfere with the measurement of the sample. May be coated with a sheet of sex (27). An example of such an impermeable sheet is cellophane. Other than the overlapping portion of the hemolytic agent-carrying layer (25) can not be coated with the impermeable sheet. A blood sample is added to the uncoated portion. obtain. The impermeable sheet (27) is at least a part of the overlapping region of the antibody-immobilized membrane (22) and the absorbing layer (26) and the non-overlapping absorbing layer (26) of the antibody-immobilized membrane (22) Can also be coated. Such a coating does not interfere with the absorption from the antibody immobilization membrane (22) to the absorption layer (26). As described above, when measuring the components in blood cells, the dilution step which has conventionally been necessary can be eliminated by partially lysing the blood sample. In the case of manual operation, the dilution operation can be omitted by using the method of the present invention. Also, even if the dilution step has been automated up to now, the automatic dilution step can be omitted from the automation equipment, which leads to downsizing of the device and cost reduction.

 Therefore, according to the present invention, a blood processing method, a hemoglobins measuring method, a blood processing device and a hemoglobins measuring device, which can more easily measure components in blood cells such as hemoglobins, are provided. According to the present invention, hemoglobins can be measured simply and quickly. Example

Next, an embodiment of the present invention will be described based on a measurement example of HbA _lc . The present invention is not limited at all by the following examples. Materials, reagents and the like used in the examples are obtainable from commercial sources, unless otherwise specified.

(Example 1: Appropriate range of addition amount of hemolytic agent in absorption method)

To that of potassium chloride 1.5, 7.5, 15, 75, and 150 mg, whole blood sample (HbA 1 c value 4.8%) 1.5 ml was added (ie, it it potassium chloride 0.1 %, 0.5%, 1%, 5%, and 10%; these proportions are weight / volume), cloudy, and then centrifuged to remove 1.0 ml of supernatant fluid. The results of examining the light transmittance at 350 to 800 nm for each of these supernatants are shown in FIG. For example, at 500 nm, the addition amount of the hemolytic agent (here, KC 1) is suitably about 0.1 to 0.5%, and for 700 nm, about 0.1 to 10% is appropriate. Is an indicator of (Example 2: Amount of hemolytic agent, 1113 concentration curve 11 _{1 (} relationship with value)

 Whole blood sample (Hb Al e value 4. 8%) was added to 1.5, 7.5, 15, 75, or 150 mg of potassium chloride. 1%, 0.5%, 1%, 5%, or 10%; these proportions are weight / volume), clouded and then centrifuged, and 1.0 ml of supernatant fluid was removed. The concentration of Hb present in the supernatant was determined by S L S-hemoglobin method. S L S—Describe the hemoglobin method. The SLS-hemoglobin method was performed using Hemoglobin B-Test, which is commercially available from Wako Pure Chemical Industries, as a dedicated reagent. Add 0.12 ml of hemoglobin standard solution (1, 5, 10, 50, 100, or 150 g / L) to 0.50 ml of 0.67 mM sodium laurate, which is a chromogenic stock solution of hemoglobin, and mix well Then, it was allowed to stand at room temperature for about 3 minutes to prepare a hemoglobin control solution. The absorbances of these control solutions were measured at a wavelength of 540 η m. Using the obtained values, a calibration curve of absorbance (540 nm) was prepared for hemoglobin concentration. Next, in samples which had been treated with various hemolysis rates, the absorbance was similarly measured using 0.02 ml of the above-mentioned supernatant fluid. Each hemoglobin concentration was calculated using a calibration curve.

Meanwhile, Hb A ,. The values were measured using a dedicated HP LC (Al e 2.2, manufactured by Tosoh 1). The hemolyzed sample was placed in a test tube, set in this device, and measured automatically. The Hb value obtained here corresponds to the ratio (%) of HbA _{lc to} total hemoglobin.

The above results are shown in Fig.7. In FIG. 7, the lower horizontal axis shows the concentration (%) of potassium chloride added, the left vertical axis shows the Hb concentration (mg / ml), and the right vertical axis shows the HbA _lc value ( %) Is shown. Also, black circles in FIG. 7 indicate the results of Hb concentration, and white circles indicate the results of Hb A _lc values.

FIG. 7 shows that depending on the KC1 concentration, the Hb concentration by hemolysis is increased. Furthermore, depending on the amount of hemolytic agent, Hb concentration is changed, but HbA value is not changed. Therefore, there is no need for complete hemolysis in the determination of the percentage of hemoglobins to total hemoglobin, thus the need for a dilution step. It was suggested that the need would disappear.

Example 3 Measurement of HbA 1 c Using Immunochromatography

 (2. 1. Preparation of Device for Measuring Hb and Device for Measuring HbA 1 c) In this example, a device that can be used for measuring Hb A 1 c was manufactured. This device, as shown in FIG. 5, is a multilayer system in which hemolysis by the hemolytic agent, reaction with the antibody, and detection of this reaction are performed in separate layers. FIG. 5 is a cross-sectional view of an example of the multilayer device of the present invention. First, preparation of each layer will be described below. Gold Colloid-Labeled Antibody Lyophilized Immobilized (24)>

In an Erlenmeyer flask, 198 ml of distilled water and 2 ml of a 1% aqueous solution of chloroauric acid were added, the obtained mixed aqueous solution was boiled, and 4 ml of a 1% aqueous solution of citric acid and a 3% aqueous solution of sodium hydroxide was rapidly added to the boiled aqueous solution. After addition, after confirming that the color of the mixed aqueous solution turned to reddish purple, it was left to stand at boiling for 15 minutes. After that, the Erlenmeyer flask was taken out, and left to stand at room temperature to cool naturally to obtain a gold colloid solution. An aqueous 0.2 M potassium carbonate solution was added to the obtained gold colloid solution to adjust to pH 8.9. A solution of 1.55 mg / ml of a solution of anti-Hb monoclonal antibody produced from the cell line of National Institute of Advanced Industrial Science and Technology, International Patent Organism Deposited at Accession No. FERM BP-7288 for this gold colloid solution is used. Add and let stir for 3 minutes. Further, 20 ml of 10% aqueous solution of serum albumin (BSA) (pH 8.9) was added, and the mixture was further stirred for 3 minutes. Thereafter, the mixture was centrifuged to remove unlabeled antibody and BSA, and the supernatant was removed by vortex. For washing, the cells were resuspended with a solution of 1% serum serum albumin-saline (BSA · PBS) solution, centrifuged again, and the supernatant was removed by vortex. The gold colloid-labeled anti-Hb monoclonal antibody 1% BSA · 卩 83 suspension thus obtained was filtered using a 0.45 / m filter to remove aggregates. After that, a glass fiber filter paper was impregnated with a suspension of gold colloid-labeled anti-Hb antibody 21 and frozen using liquid nitrogen, and left in a lyophilizer for 24 hours to prepare a freeze-dried immobilized product. Hb measurement / Immobilizing antibody (22)>

 A solution of anti-Hb monoclonal antibody (PBS) produced from the cell line of National Institute of Advanced Industrial Science and Technology, International Patent Organizing Center for Patent Organisms, Accession No. FERM BP-7289 was immobilized on a nitrocellulose membrane. After immobilization, blocking was done with skimmed milk. Subsequently, the immobilized antibody was washed twice with 0.1 M Tris (pH 8.2) and dried. Antibody-immobilized membrane for Hb measurement (22)>

 A solution of an anti-H b A 1 c monoclonal antibody produced from the cell line of National Institute of Advanced Industrial Science and Technology, International Patent Organizing Center for Patent Organisms, Accession No. FERM BP-7636 was immobilized on a nitrocellulose membrane. After immobilization, blocking was done with skimmed milk. Subsequently, the immobilized antibody was washed twice with 0.1 M Tris (pH 8.2) and dried. Hemolytic agent loaded solid phase (25)>

 Lead Cooking Paper (registered trademark) (sold by Lion Corporation) was impregnated with 0.5% (w / v) KC 1 based on the total sample volume and lyophilized.

<Construction of device for H b measurement and device for H b A measurement>

Gold colloids labeled antibody lyophilized carrying layer produced above, by using the Hb measurement antibodies fixed turtle Nburen or Hb A _lc measuring antibody immobilized membrane, and hemolytic agent carrying layer, as shown in FIG. 5, Hb The device for measurement and the device for Hb A measurement were built. The construction of this device is described below. Antibody-immobilized membrane (22) on which antibody (23) is immobilized on a substrate (21) (polyethylene terephthalate: manufactured by Matsumoto Seisakusho), followed by freeze-drying of gold colloid labeled antibody on this. The layer (24) and subsequently the hemolytic agent-carrying layer (25) were laminated thereon. Here, as shown in FIG. 5, a hemolytic agent-supporting layer (25), a gold colloid-labeled antibody freeze-drying-supporting layer (24), and an antibody-immobilized membrane (22) Was placed to overlap the layer immediately below. The antibody-immobilized membrane (22) is specific for hemoglobins in the bare area (ie, the area where the hemolytic agent-carrying layer (25) and the gold colloid-labeled antibody lyophilised carrier (24) do not overlap). This region to which the antibody was fixed was used as a region for detecting the reaction of hemoglobins in the blood sample sample. Also, on the antibody-immobilized membrane (22), overlap the area on the opposite side to the blood sample sample addition side (that is, the side where the gold colloid-labeled antibody lyophilization support layer (24) is not overlapped). A glass fiber filter paper absorbent layer (26) was laminated. In addition, the reaction detection area from the overlapping area of the hemolyzing agent-carrying layer (25), the gold colloid-labeled antibody lyophilization-carrying layer (24), and the antibody-immobilized membrane (22) is shown in FIG. ) (Cellophane) coated. The portions other than the overlapping portion of the hemolytic agent-carrying layer (25) were exposed. This exposed area is the area where the blood sample is spotted. The impermeable sheet (27) is a glass fiber filter paper absorbent layer in which the overlapping area between the antibody immobilized membrane (2 2) and the glass fiber filter absorbent layer (26) and the antibody immobilizing membrane (22) do not overlap ( 26) also covered a part of. This is to prevent interference with absorption from the antibody-immobilized membrane (22) to the glass fiber filter paper absorbent layer (26).

<Measurement of hemoglobins using a device for Hb measurement and a device for Hb A _lc measurement>

 0, 0. 05, 0. 1, 0. 5, 0, 5, 0, 7, 0, 0, 0, 0, 0, 0, 0, 7, 0, 0, 0, 0, 0, 0, 0, 0, 7, 0, 0, 0, 0. 0 In contrast, 160 μl of blood was spotted, and after 5 minutes, the reflection absorbance at 610 nm was measured. The results are shown in Fig.8.

As shown in FIG. 8, by showing the coloration intensity at 0.1 to 1.0% (w / v) KC 1, the optimum range of the amount of KC 1 is 0.1 to 1.0% (w / v). ) It can be seen that it is. In this device, a half value of 0.5% (w / v) KC1 is preferable, and as described above in the section of <Hemolytic agent-supporting solid phase (25)>, in this example, 0.5% (w / v) KC1 was supported on a hemolytic agent-supported solid phase (25), and an attempt was made to measure HbA _lc . First, dilution series were prepared using Hb standard solution or HbA _lc standard solution. The dilution series 1 60 / L was spotted on the device, and after 5 minutes, the reflection absorbance at 6 10 nm was measured to prepare a calibration curve. The calibration curve of the device for Hb measurement is shown in Fig. 9, and the calibration curve of the device for HbA measurement is shown in Fig. 10. In FIG. 9, the abscissa represents the Hb concentration (mg / d 1), and the ordinate represents the value of the reflection absorbance at 61 nm. Also,

Black circles in 9 indicate the results of absorbance of Hb concentration in each dilution series. In FIG. 10, the abscissa represents the Hb A _lc concentration (mg / d 1), and the ordinate represents the value of the reflection absorbance at 6 10 nm. Also, the black circles in FIG. 10 indicate the results of the absorbance of the Hb A _lc concentration in each dilution series.

Next, 60 l of a blood sample of 1 containing 60% of 4.0, 5.4, or 10. 4% 11 _1. 8 ₍ : :) was spotted onto the device, and after 5 minutes, the reflection absorbance was measured. in Figure 1 1 are shown. FIG. 1 1, HbA value on the horizontal axis indicates (%) shows the reflection absorbance for Hb to the left vertical axis indicates the reflection absorbance for HbA _lc on the vertical axis on the right. Further, FIG. The black circles in 11 indicate the results of Hb concentration, and the white circles indicate the results of HbA _lc values.

Each absorbance value of the 11 results was regressed from the calibration curve obtained from FIG. 9 and FIG. 10 respectively to determine the Hb concentration and the HbA _lc concentration (FIG. 12). In FIG. 12, the abscissa represents the reflection absorbance at 6 10 nm, and the left ordinate represents the H b concentration calculated by conversion.

shows (mg / d 1) and the HbA concentration calculated by conversion on the right vertical axis

(mg / d 1) is shown. The black circles in Fig. 12 indicate the results of Hb concentration, and the white circles indicate the results of HbA concentration (mg / dl). The Hb concentration was almost the same in all of the above blood samples. From this, it is understood that the above-described blood samples are hemolyzed to the same extent. Using the Hb concentration obtained by the measurement on the device (mg / dl) and HbA _lc levels (mg / d 1), Hb A lc values of samples of each HbA _lc value (%) was determined (HbA _lc conc Degree (mg / d 1) / Hb concentration (mg / d 1) x 100 calculated). As a result, HbA ,. Samples with a value of 4.0% were calculated to be _3.9 %, samples with an HbA value of 5.4%, and samples with an HbA _lc value of 10.4% were calculated to be 5. 12%. From this, it has been confirmed that hemoglobins can be measured by controlling to partially hemolyze blood sample samples. From the above results, it was possible to create a device that can be easily measured in one step without the dilution step.

 According to the present invention, hemoglobins can be measured simply and rapidly. Industrial applicability

 The present invention is useful in the field of nutrition management and medical diagnostics.

Claims

The scope of the claims

 1. a step of preparing a blood sample containing red blood cells and a hemolytic agent (a),

 Preparing a mixed sample by mixing the blood sample and the hemolytic agent, and (b)

 The hemolytic agent is a solid, which is a drug that destroys an erythrocyte membrane.

2. In the blood processing method according to claim 1,

 In the step (b), a blood cell component remains in the mixed sample.

3. In the blood processing method according to claim 1,

 The hemolytic agent is a drug that changes the osmotic pressure to the erythrocyte membrane.

4. In the blood processing method according to claim 3,

 The hemolytic agent is an inorganic salt.

5. In the blood processing method according to claim 4,

 The blood processing method, wherein the concentration of the inorganic salt in the mixed sample is in the range of 0.50 to 10% (w / v).

6. Preparing a blood sample containing red blood cells and a hemolytic agent (a)

 Preparing a mixed sample by mixing the blood sample and the hemolytic agent (b);

 And (c) measuring hemoglobins contained in plasma in the mixed sample,

The above-mentioned hemolytic agent is a solid. Hemoglobins measuring method which is a drug which destroys an erythrocyte membrane.

7. In the method for measuring hemoglobins according to claim 6,

 In the step (c), as a method of measuring hemoglobins contained in plasma in the above mixed sample, an immunochromatography method, an immunoconcentration method, an enzyme-linked immunosorbent assay method, a colorimetric slide method, and A method of measuring hemoglobins in which any one of the electrode method slide methods is used.

8. In the method for measuring hemoglobins according to claim 6,

 A method for measuring hemoglobins, wherein at least two types of hemoglobin are measured in the step (c).

9. In the method for measuring hemoglobins according to claim 8,

 The method for measuring hemoglobins, wherein the at least two types of hemoglobin are a combination of hemoglobin and H b A 1 c.

In the method for measuring hemoglobins according to claim 6, 10

 The above hemoglobins are glycated hemoglobins.

1 1. With a substrate

 Equipped with a solid hemolytic agent to destroy the erythrocyte membrane,

 The hemolytic agent is carried on the substrate in such an amount that the blood cell component remains in the mixed sample when the mixed sample of the blood sample containing red blood cells and the hemolytic agent is formed. Blood processing device.

1. A hemoglobin measuring device for measuring hemoglobins in a blood sample containing red blood cells,

 A substrate having the addition part of the above blood sample,

 A hemolytic agent for destroying the erythrocyte membrane,

 A detection substance that specifically binds to the hemoglobins;

An immobilized substance that specifically binds to the above-mentioned hemoglobins; The hemolytic agent is carried on the substrate in such an amount that the blood cell component remains in the mixed sample when the mixed sample of the blood sample and the hemolytic agent is formed; The detection substance and the immobilization substance are each solid and supported on a substrate,

 The hemoglobin measurement device, wherein the hemolytic agent, the detection substance, and the immobilization substance are arranged in order from the side closer to the addition part of the blood sample of the base material.

A hemoglobin measuring device for measuring hemoglobins in a blood sample containing red blood cells, the device comprising:

 A substrate,

 An addition part of the blood sample provided on the substrate;

 A hemolytic layer provided on the substrate, comprising a solid hemolytic agent for destroying the erythrocyte membrane, and controlling the hemolysis of the blood sample sample;

 A labeling layer provided on the substrate and containing a detection substance that specifically binds to the hemoglobin, and for labeling the hemoglobin;

 And a detection layer for detecting the hemoglobins, comprising an immobilization reagent provided on the substrate and specifically binding to the hemoglobins.

 The hemoglobins measuring device, wherein the blood sample sample can move between the hemolytic layer, the labeling layer, and the detection layer.

The device for measuring hemoglobins according to claim 14.

 A hemoglobin measurement device, further comprising a layer for removing red blood cell residue after the blood sample is hemolyzed.

The device for measuring hemoglobins according to claim 13

 The hemoglobins measuring device, wherein the hemolytic layer, the labeling layer, and the detection layer are disposed in a single layer.